The invention disclosed herein was made with Government support under NIH Grant Nos. CA-31397 and CA-56909 from the Department of Health and Human Services and the Department of Energy OHER (DC). Accordingly, the U.S. Government has certain rights in this invention.
Within this application, publications are referenced within parentheses. Full citations for these references may be found at the end of each series of experiments. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
Two distinct processes involving DNA double-strand breaks (DSB) have been identified in mammalian cells: the repair of DNA damage induced by ionizing radiation and V(D)J recombination during T- and B-cell development. So far, all mammalian cell mutants defective in DNA DSB repair share the common phenotype of hypersensitivity to radiation, and impaired ability to undergo V(D)J recombination (1-6). Cell fusion studies using DSB repair mutants of human-rodent somatic hybrids have defined four complementation groups: IR4, IR5, IR6, and IR7. Genetic and biochemical analyses have revealed that cells of IR5 (e.g., xrs-6) and IR7 (e.g., scid) are defective in components of the DNA-dependent protein kinase (DNA-PK) (2, 7-9). DNA-PK is a serine/threonine kinase comprised of a large catalytic subunit (DNA-PKcs) and a DNA-targeting component termed Ku, which itself is a heterodimer of a 70-kDa (Ku70) and a 86-kDa (Ku80) polypeptide (10-12). Recently, DNA-PKcs has been shown to be the gene responsible for the murine scid (severe combined immunodeficiency) defect (13-15); and Ku8O has been identified to be XRCC5 (16-18), the X-ray-repair cross-complementing gene for IR5. Ku80 knockout mice were found to exhibit severe combined immunodeficiency, defective processing of V(D)J recombination intermediates, and growth retardation (19, 20).
Though Ku70 has been designated as XRCC6 (7, 8) and is an important component of the DNA-PK complex, the function of Ku70 in vivo is hitherto unknown. To define the role of Ku70 in DNA repair and V(D)J recombination, we targeted the Ku70 gene in mice. Ku70 homozygotes exhibit proportional dwarfism, a phenotype of Ku80xe2x88x92/xe2x88x92, but not of scid mice. Absence of Ku70 confers hypersensitivity to ionizing radiation and deficiency in DNA DSB repair, which are characteristics of both Ku80xe2x88x92/xe2x88x92 and scid mice. Surprisingly, in contrast to Ku80xe2x88x92/xe2x88x92 and scid mice, in which both T- and B-lymphocyte development are arrested at early stage, lack of Ku70 is compatible with T cell receptor gene recombination and the development of mature CD4+CD8xe2x88x92 and CD4xe2x88x92CD8+ T cells. Our data, for the first time, provide direct evidence supporting that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR gene recombination. These results suggest that distinct but overlapping repair pathways may mediate DSB repair and V(D)J rejoining; furthermore, it suggests the presence of a Ku70-independent rescue pathway in TCR V(D)J recombination. The distinct phenotype of Ku70xe2x88x92/xe2x88x92 mice should make them valuable tools for unraveling the mechanism(s) of DNA repair and recombination. Ku is a complex of two proteins, Ku70 and Ku80, that functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase (DNA-PK). The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation and V(D)J recombination was examined in mice that lack Ku70 (Ku70xe2x88x92/xe2x88x92). Like Ku80xe2x88x92/xe2x88x92 mice, Ku70/xe2x88x92 mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80xe2x88x92/xe2x88x92 mice, in which both T- and B-lymphocyte development were arrested at early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4+CD8xe2x88x92 and CD4xe2x88x92CD8+ T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.
This invention provides a method of diagnosing a predisposition to cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and; (b) determining whether one or more of the subject""s Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject""s expression of polypeptide having tumor suppressor activity.
This invention also provides a method of assessing the severity of cancer in a subject comprising: (a) obtaining a nucleic acid sample from the subject; and (b) determining whether one or more of the subject""s Ku70 alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce or eliminate the subject""s expression of polypeptide having tumor suppressor activity.
This invention also provides a method of assessing the severity of cancer in a subject comprising: determining the subcellular localization of Ku70 in the subject, wherein an abnormal subcellular localization of Ku70 indicates a predisposition to cancer.
In addition, this invention provides the above-described methods, wherein the abnormal subcellular localization of Ku70 comprises increased cytosolic localization of Ku70.
In addition, this invention provides a method of inhibiting the growth of cancer cells, comprising introducing into a cell a Ku70 gene under conditions permitting expression of the gene.
This invention also provides the above-described methods, wherein the cancer is T-cell lymphoma.
In addition, this invention provides the above-described methods, wherein the cell prior to the introduction of the Ku70 gene was characterized as having a mutation at one or more Ku70 alleles or regulatory regions thereto.
In addition, this invention provides the above-described methods, wherein the cell prior to the introduction of the Ku70 gene was characterized as having reduced expression of Ku70.
This invention further provides the above-described methods, wherein the Ku70 gene is incorporated into an expression vector prior to introduction into the cell.
This invention also provides a method of inhibiting the growth of cancer cells, comprising introducing Ku70 into a cell.
In addition, this invention provides the above-described methods, wherein the cancer is T-cell lymphoma.
In addition, this invention provides a transgenic cell, wherein the expression of the Ku70 allele have been altered to increase the susceptibility of the cell to DNA damage.
This invention also provides a transgenic cell, wherein the expression of the Ku70 allele has been altered to increase the susceptibility of the cell to cancerous growth.
This invention also provides a transgenic organism, comprising an organism whose germ line cells has been altered at the Ku70 allele to produce an organism whose offspring have an increased likelihood of developing tumors.
In addition, this invention provides a transgenic organism, comprising an organism whose germ line cells has been altered at the Ku70 allele to produce an organism whose offspring have an increased likelihood of having increased susceptibility to DNA damage.
This invention further provides a method of screening a compound for carcinogenic activity, comprising: (a) contacting cells having reduced expression of Ku70 with the compound; and (b) determining whether the compound results in a malignant transformation phenotype.
This invention also provides a method of screening a compound for ability to restore Ku70 activity to cells having Ku70 defect symptoms resulting from reduced Ku70 activity, comprising: (a) contacting cells having reduced expression of Ku70 with the compound; and (b) determining whether the compound restores, in whole or in part, a normal Ku70 phenotype.